Abstract/ Project Summary Immune checkpoint inhibitors used to treat patients with cancer can enhance adaptive anti-tumor immunity and are remarkably efficacious, but produce durable responses on only 10-15% of patients, warranting additional studies of tumor immunity. Little is known about how cancers might respond to treatments that activate intrinsic immunity, the collection of molecular pathways present in nearly all cells that provide first-line recognition of, and protection against, viruses. Activation of endogenous viral oligonucleotide receptors, such as RIG-I, could be used therapeutically as a way to jumpstart an otherwise immunosuppressed tumor microenvironment (TME). Once RIG-I binds to viral RNA, RIG-I signaling potently induces activity of pro- inflammatory transcription factors IRF3 and NF-?B, activating expression of pro-inflammatory cytokines. Simultaneously, RIG-I activates the caspase-1 dependent inflammasome, which initiates an immunogenic form of programmed cell death termed `pyroptosis.' In the cancer setting, pyroptosis in combination with pro- inflammatory cytokines could be therapeutically efficacious. Although RIG-I signaling has not been well-studied in breast cancers, a cancer type often considered immunologically `cold,' my preliminary data shows that breast cancer cells treated with a synthetic RIG-I mimetic undergo cell death, and produce an abundance of the pro-inflammatory cytokine interferon (IFN)-? and TRAIL. Further, we have used the RIG-I mimetic to treat mouse mammary tumors in vivo, finding decreased tumor growth and increased tumor cell death, consistent with the hypothesis that activation of intrinsic immunity in breast cancers will induce pyroptosis, expression of pro-inflammatory cytokines, and increase tumor infiltrating lymphocytes. We propose three Aims to test this hypothesis. Aim 1 will focus on the molecular mechanism(s) by which RIG-I signaling induces cell death in breast cancer cells. Aim 2 will identify cytokines induced by RIG-I in breast cancer cells, and the molecular pathways regulating their expression downstream of RIG-I activation. Aim 3 will focus on how therapeutic RIG-I signaling impacts mammary tumor leukocyte populations in vivo, as well as how RIG-I signaling effects immune checkpoint inhibitor therapy in breast cancer. We will employ a combination of detailed molecular analyses, unbiased molecular arrays, mass cytometry (CyTOF), and intravital imaging to question how RIG-I activation might impact disease progression in breast cancers.